Method of producing d-ribose

ABSTRACT

D-ribose is produced by cultivating a strain belonging to the genus Bacillus whose transketolase activity is nil to cause said strain to elaborate and accumulate a large amount of D-ribose. The thus accumulated D-ribose can be recovered in a good yield.

United States Patent [191 Sasajima et a1.

[ METHOD OF PRODUCING D-RIBOSE [75] Inventors: Ken-ichi Sasajima, Hyogo;

Masahiko Yoneda, Kobe. both of Japan {731 Assignee: Takeda ChemicalIndustries, Ltd.,

Osaka, Japan [22] Filed: Nov. 14, 1973 [21] Appl. No: 415.561

Related US. Application Data 163] ContinuationJn-parl of Ser. No.370759. June 18.

1973 abandoned,

[30] Foreign Application Priority Data June 17. 1972 Japan 4160612 [52]US. Cl 195/30; 195/29; 195/28 R;

1 1 NOV. 11,1975

Primary ExuminerAlvin E. Tanenholtz AIIUI'IM), Agenl or FirmWenderoth.Lind & Ponack {57] ABSTRACT D-ribose is produced by cultivating a strainbelonging to the genus Bacillus whose trunsketolase activity is nil tocause said strain to elaborate and accumulate a large amount ofD-ribose. The thus accumulated D- ribose can be recovered in a goodyield 11 Claims, N0 Drawings METHOD OF PRODUCING D-RIBOSE Thisapplication is a continuation-in-part of Ser. No. 370.759. filed .luneI8, 1973, now abandoned.

This invention relates to a method of producing D- ribose. By thepresent invention is provided a method of producing D-ribose. whichcomprises cultivating a D-ribose-producing microorganism belonging tothe genus Bacillus whose transketolase activity is nil in a culturemedium containing assimilable carbon and nitrogen sources as well asfactors necessary for the growth of the strain. thereby causing saidstrain to elaborate and accumulate D-ribose and, then, recovering theD-ribose thus accumulated from the resultant culture broth.

As a constituent of nucleic acids. D-ribose occurs in all organisms. andribitol. a reduction product of D- ribose. is present in vitamin B andribitol-teichoic acid. a constituent of cell walls. Thus, it is a veryimportant substance physiologically. Furthermore, D-ribose and itsderivatives have so far attracted a great deal of attention as startingmaterials for the synthesis of vitamin B and the so-called nucleic acidcondiments and. accordingly; the development of a commercial process forthe production of D-ribose has been much desired.

The conventional methods for producing D-ribose include methods forextracting D ribose from natural products and synthetic methods usingfuran. D-glucose. etc., as starting materials. There also are reports onthe ,fermentative production of D-ribose. but the fennentation yield isextremely low. Thus. these processes are not fully satisfactory ascommercial processes for the production of D-ribose.

The present inventors previously disclosed methods of producing Dribosecomprising utilization of Bacillus organisms.

These methods are concerned with the use of a D- ribose-producingmicroorganism of the genus Bacillus which requires L-tyrosine.L-tryptophan and L- phenylalanine for its growth (hereinafter, themicroorganisms mentioned above are collectively referred to as the aminoacids-requiring microorganisms") or which lacking in transketolaseactivity (hereinafter, these microorganisms are referred to as thetransketolaselacking microorganisms).

The former method in which are used the amino acids-requiringmicroorganisms is described in the specification of British Pat. No.l,255,254 and the latter in which are used the transketolase-lackingmicroorganisms is described in Agricultural and Biological ChemistryVol. 35. No. 4. page 509 (1971).

The amount of D-ribose obtained by these methods ranges from about mg.to about mg. per ml. of the fermented broth.

Both the amino acids-requiring microorganisms and thetransketolase-lacking microorganisms have extremely low transketolaseactivity, especially the latter microorganisms were considered aslacking in transketolase, on the basis of the experimental results onthe activities measured by the method of B. L. Horecker et al. [Journalof Biological Chemistry Vol. 223. page I009. 1956)] described below.

1. Preparation of the Crude Enzyme Solution The slant culture of astrain whose transketolase activity is to be determined is transferredinto 200 ml. of a modified Spizizen's medium [Agricultural and Biological Chemistry Vol. 34. No. 3 page 38l l970)] supplemented withshikimic acid. containing 0.4% of (NHQ SO 1.4% of K HPO 0.6% of KH PO0.02% of MgSO .7H O. 0.0004% of biotin, 0.00687: of ade nine. 0.014% ofguanosine. 0.0l% of shikimic acid. 0.5% of sorbitol and 0.5% of sodiumL-glutamate as carbon sources in a l-liter flask and incubated at 37C ona rotary shaker. After l6-hour cultivation. the cells are harvested bycentrifugation. washed with 0.01 M tris-(hydroxymethyl)aminomethane-HCIbuffer solution (pH 7.5) containing 0.00l M mercaptoethanol andsuspended in the same buffer solution so that the absorbancy at 650 muof the cell suspension may be 10. Then. lysozyme is added to the cellsuspension at a concentration of 50 ug. per ml. The suspension isincubated at 37C for min. The cellular debris are removed bycentrifugation (l.2 l0g). The clear solution is used as the enzymesolution.

ll. Preparation of Reaction Solutions Reaction Solution A 20 ,u moles ofD-ribose S-phosphate, 0.5 p. mole of NADH. D-ribose S-phosphateisomerase. D-ribose 5- phosphate epimerase, 0.66 unit ofa-glycerophosphate dehydrogenase containing triosephosphate isomeraseand 60 u moles of tris( hydroxymethyl )aminomethane- HCl buffer solution(pH 7.5). Reaction Solution B 20 IJ. moles of MgCl 0.43 t mole ofthiamine pyrophosphate. 40 pt moles of tris(hydroxymethyl-)aminomethane-HCI buffer solution (pH 7.5 a given amount of the enzymesolution.

lll. Assay of Transketolase Activity Each reaction solution is incubatedat 30C for 10 min. and then the reaction solutions are mixed (the totalvolume is 2 ml. and its absorbancy at 340 my. was measured with thelapse of time.

The transketolase activity (p. molelminlmgprotein) in the enzymesolution is expressed by the following formula:

wherein AE is the velocity of decrease of the net absorbancy of themixed reaction solution at 340 mu for 1 minute. V is the total volume ofthe mixed reaction solution (2 ml.), E is the molecular extinctioncoefficient of NADH at 340 mu (6.22 cmF/p. mole). d is the light path (lcm), E is the volume of the enzyme solution (0.l ml.), and p is theweight of protein in the enzyme solution (mg/ml).

As the test organisms were employed known strains of the aminoacids-requiring microorganisms. i.e.. Bacillus pmm'lus No. 503 and No.537 and Bacillus subrilis No. 429 (British Pat. No. l.255.254) as wellas those of the transketolase-lackin g microorganisms. i.e.. Bacillusspecies Shi 5 and Shi 7 (Agricultural and Biological Chemistry Vol. 35.No. 4 page 509 I971 As the organisms to be compared were employed wildstrains of Bacillus pmnilus and Bacillus sublilis.

The results are shown in Table l.

Table l transprotein optical ketulase Organism (mg/mil density acti\ itl \E1mr (u molesl l'l) min.) min/mgv protein) Bacillus pumilm 2.1 0. I650.24 I

(\iild strain] Bmillus pumilus' l.'-) (Lllll 0.00 (I No. 503 Bucillmpumilus L8 l).l)()5 (Hill 0 No. 537 Butlllus .iuhlilis 2.!) 0.080 (H3lllU l\\llll strain) Bm'illui .\ul1lill.\ l.7 U.UU5 (Lilli l) No. 439Bacillus spar ic.\ 1.8 ().t)(l5 0.00 0

Shi 5 Bacillus species [.9 (J.(Jl|$ 0.00 t) Shi 7 In the above assay ofthe transketolase activity, for the enzyme solution was employed theclear solution as it was, and the activity was found to be nil at leastdown to two decimal places. But no significant figure at the thirddecimal place was obtained by this method.

cry that strains whose transketolase activity is nil at least down tothe third decimal place are capable of accumulatin g a higher amount ofD-ribose than the amino acids-requiring microorganisms and thetransketolaselacking microorganisms and can accumulate D-ribose in anamount of about mg. to about mg. per ml. of the culture broth obtainedby the same manner as the known methods. it was further found that thestrains whose transketolase activity is nil at least down to the thirddecimal place (hereinafter simply referred to as the Microorganisms")accumulate a much greater amount of D-ribose, when they are cultivatedin the presence of a dibasic organic acid. This invention is theculmination of the above findings.

The microorganisms employed in the practice of this invention can beobtained by using such wild strains as Bacillus pumilus, Bacillussubiilis, etc, as parent strains and subjecting them to radiation suchas ultraviolet light, X-rays or 'y-rays or a treatment with a mutagensuch as nitrosoguanidine. Examples of the strain to be exploited in thepractice of this invention include Bacillus pumilus No. 716, No. 735 andNo. 783 and Bacillus subrilis No. 608 and No. 632. Table 2 shows thetran sketolase activities of these strains in comparison with Furtherstudlet' on the of the trmsketolasc 35 Bacillus pmnilus No. 503 and No.537 and Bacillus subxp led to elopment 0f the l b} Iilis No. 429,Bacillus species Shi 5 and Shi 7, employing IC the calculation of thetransketolase activity can the enzyme solution as it is (method D Andemploying be made correctly down to the third decimal place on theconcentrated enzyme Soluion (mehod m Table 2 Relathe transketolaseactivities Organism Method l Method ll protein ahsorhancy transketolaseprotein absorbancy transketolase (mg/ml.) (.\E=uiw..m.l acti\ itv(mg/ml.) (.\E activitv (u moles/min] (/1) min.) (u moles/min]. mg.protein) mg. protein) Bacillus 1mm ilm (Wild strain) 3.: (1.24 lllO lUUBacillus pumilux N0. 503 1.9 (LUU5 (Llltl I (J.(lll l9.l 0.030 0.005 IBacillus pumilm NO. 537 ill ll.tlil5 Ull'l) (Lill) (l l9. (LU-l5 (LOOK 3Bacillus pmuilus NO. 7H1 1U ll.ll(l5 (MK) U.lll (I 10.3 U.(JU5 (Milli)ll.llill) U Bncillm pumilm N0. 733 2. ().()(l. (Mill O.()l] H 23.0U.tlll5 ()llllll ().()(ll l Bacillus .rulnilis (wild strain} 3.0 0.080(H3 I00 I00 Bacillus .vulnilix N0. 429 I7 ll.i|ll5 llllll I U.Ul l O20.0 0.025 (1.004 3 Barillux suhiilis NO. 603 2.1 U.tl()5 (L00 (Hll U214 ll.(lll$ 0.000 ().()lll Bacillus .mhlilis N0. 632 2.] (HKJ5 llllilll.lll) 22.1 ().U(l5 ODUU (l.(l(ll) ll Bacillus .i'pcrit'x Sl'll 5 L8l).()(l5 ().()(l (l.lll] l) I95 (LU-ill (1.007 3 Bacillus spcciav Shi 71.) ().()U5 0.01) ().()l i911 (1.045 (L007 3 a concentrated solutionobtained by ultrafiltrating the enzyme solution in the above-mentionedassay method. That is. the enzyme solution was concentrated by theultrafiltration method using ultrafiltration membrane, for exampleDlAFLO membrane PM 10 (trade mark of Amicon Corporation. U.S.A.), untilits volume becomes about one-tenth of its original volume. And thepresent inventors used thus obtained concentrated solution as the enzymesolution.

This method made it clear that the transketolase activity of the aminoacids-requiring microorganisms and of the transketolase-lackin gmicroorganisms was not nil at third decimal place. Further improvementstudies on strains and cultivation methods have led to the discov- Twomilliliters of the seed culture was transfered to ml. of a main culturemedium (hereinafter called Medium A) composed of 15% of D-glucose, L57:of dried yeast. 0.5% of ammonium sulfate, 2% of calcium carbonate, 100y/ml. of L-tryptophan, l00 'y/ml. of L- phenylalanine and 100 'y/ml. ofL-tyrosine in a 200 ml. creased flask and incubated at 37C for 6 days ona rotary shaker at 200 rpm.

Amount of D-ribose accumulated in a culture broth was determined by themethod described in Agricultural and Biological Chemistry, Vol. 35, page509 197) Table 3 Relative rihose-producing abilities D-rihoseaccumulated The genetic characters of the microorganisms are that theycannot grow on such carbohydrates as D-gluconic acid, L-arabinose andD-ribose as exclusive carbon sources and they require for their growthshikirnic acid or any substitutive substances such as L-tyrosine.L-tryptophan or L phenylalanine.

Other genetic and biochemical characters of these strains are that incontrast to their parent organisms and to the known strains, theirtransketolase activities are below the presently known threshold ofdetection and their elaboration and accumulation of D-ribose areconsiderably high.

In cultivating the Microorganisms, one may use, as carbon sources,D-glucose, D-fructose, D-mannose, sorbitol, D-mannitol, sucrose,maltose, dextrin. soluble starch, spent molasses, etc., and, as nitrogensources, various inorganic and organic nitrogenous compounds andsubstances containing them such as ammonium sulfate, ammonium nitrate,urea, corn steep liquor, dried yeast. meat extract, peptone, caseinhydrolyzate and the like.

In addition to these ingredients, it is necessary that, as factorsnecessary for their growth, shikimic acid or its derivatives (forexample, methyl ester, ethyl ester. etc.) are added to the medium.

Of course, as substitutes for shikimic acid or its derivatives. aromaticacids, i.e., L-tyrosine, L-tryptophan, L-phenylalanine and derivativesthereof (for example, methyl ester, ethyl ester, acetylate, benzoylate,etc.) may be added to the medium.

The above-mentioned aromatic amino acids need not necessarily be pureproducts. Thus, dried yeast, polypeptonc, meat extract, caseinhydrolyzate, etc, which contain aromatic amino acids can be used aswell. Therefore, when any of the above-mentioned aromatic amino acids isused as a nitrogen source. one may employ it somewhat in excess of therequirement as a nitrogen source. The accumulation of D-ribose can befurther increased by incorporating in the fermentation medium a dibasicorganic acid having 2 to 5 carbon atoms such as malic acid, oxalic acid,maleic acid, fumaric acid, succinic acid, malonic acid or glutaric acidor an alkali salt thereof, for example. Generally the proportion of suchacid is preferably within the range of 0.002 M to 0.2 M, more preferablyabout 0.003 M to 0.05 M.

Table 4 shows the effects of addition of such dibasic organic acids onthe accumulation of D-ribose when Bacillus pumllus No. 735 was employed.

Table 4 Accumulation of D-rihose (mg/ml.)

Dibacic organic Additive amount [g/l.)

acid (1. l 0.3 (1.5 l .U 3.0 4,0 5.0 (n.ll 7.0 ll).

Oxalic acid 49 48 56 55 53 55 54 40 30 [8 l5 (C O H Malonic acid 5-4 658 57 5o 53 50 50 45 (CK04H4) Succinic acid 48 53 57 5o 58 57 6 52 52 40J -I Ii) Maleic acid 50 5X 59 59 57 5 45 37 (C,O,H,)

Furmaric acid J) 56 60 57 59 k 57 5 5t) 4 (C,O,H,)

Glutaric acid 50 52 54 55 54 55 55 54 45 38 IC O H Adipic acid 48 50 4830 32 l5 l0 l0 l u 4 m) Suberic acid SI 35 Z8 l5 [7 Ill )2 H lC a -t HSebacic acid 38 2t) l5 l0 I2 I 5 5 m J IMJ Table 5 shows the typicaleffects of addition of such dibasic organic acids, when Bacillus pumllusNo. 7 l6 and Bacillus subtilis No. 608 and No. 632 were employed.

Table 5 Relation of the addition of dibasic organic acids to theaccumulation of D-ribose Accumulation of D-rihose. mg/ml.

Dibasic organic acid Bacillus Bacillus Bacillus pumilus suhtilissuhtilis Compound Amount No. No. Nov

mg./l. 716 (m8 632 Oxalic acid 0 42 45 48 (C- ,O,H- 501) 56 55 5hMalonic acid 45 45 J7 (C=,H,O,) 500 55 53 55 Maleic acid 100 45 46 42s(C,H,O.) 501) 59 57 55 Fumaric acid 500 58 55 54 (C,O,H Succinic acidNM) 46 43 49 (C.H.,O,) 500 55 52 5 Glutaric acid 100 43 46 45 (C,H,,O500 58 55 54 Adipic acid '500 45 44 37 l it i ml Suheric acid 500 27 353) [CNOJHHJ Sehacic acid 500 K ll 29 l m J ml The results shown in Table4 and Table 5 were obtained by the same culture method and assay methodas used in the experiment shown in Table 3, except that the respectivequantities of dibasic organic acids were added to the Medium A.

As will be seen from the above typical results, dibasic organic acidsare conducive to increase yields of D- ribose. and maleic acid isparticularly effective. ln addition to the above-mentioned ingredients,magnesium sulfate, calcium phosphate. calcium carbonate. etc. can befurther added.

The microorganism may be cultivated by procedures which are routinelyfollowed for the cultivation of microorganisms. although submergedculture is most expedient. While the cultivation temperature. pH andtime are largely optional. a sufficiently large amount of D-ribose iselaborated and accumulated if the strain is cultivated at to 45C and atpH 4.5 to 8.5 for about 24 to I20 hours.

The recommended procedure for recovering the D- ribose thus accumulatedfrom the fermentation broth comprises removing the cells by filtrationor centrifugation. treating the resultant filtrate or supernatant withactivated carbon and ion exchange resins to decolorize and desalt it.respectively, concentrating the same and finally adding an organicsolvent such as ethanol to the concentrate. thereby causing the desiredcompound to crystallize. In this connection. when carbohydrates otherthan the desired compound D-ribose are contaminated in the fermentationproduct. they may be removed by treating the product with glucoseoxidase or with a strain of microorganism which does not utilizeD-ribose but utilizes the particular carbohydrates.

The following examples are further illustrative of this invention. itbeing understood. however. that the invention is by no means limitedthereto.

In the present specification the abbreviation lFO shows Institute forFermentation Osaka. The percentages are weight/volume unless otherwisedescribed.

EXAMPLE I Bacillus pumilus No. 7|6 (lFO 13322] which had been obtainedby exposing the parent strain to several doses of ultraviolet radiationwas used to inoculate ID]. of a medium comprising 2% of sorbitol. 2% ofcorn steep liquor. 0.3% of dipotassium phosphate and 0.1% ofmonopotassium phosphate. The inoculated medium was incubated at 36C for24 hours to obtain a seed culture. This seed culture was then inoculatedinto llllll. of a medium comprising 15% of D-glucose. L07: of dry yeast.0.5% of ammonium sulfate, 2.0% of CaCO and 100 mg/l. of L-tryptophan andcultivated under aeration and agitation at 36C for 72 hours, whereupon45 g.lliter of D-ribose was accumulated in the medium. This D-ribosefermentation broth was filtered to remove the cells and the filtrate wasconcentrated to approximately one-half of the original volume. followedby the addition of about one quarter of its volume of ethanol. Theprecipitate was discarded and the solution was desalted with a cationand an anion exchange resin and, then. decolorized through a column ofactivated carbon. The decolorized solution was concentrated and. then.about 4 times its volume of ethanol was added. The procedure yielded 3.5kg. of crystalline D- ribose.

EXAMPLE 2 Bacillus .s'uhlilix No. 608 (IFO l33'l3) which was ob tainedby exposing the parent strain to ultraviolet radiation and. then.subjecting it to a treatment with ni trosoguanidinc. was used toinoculate lUl. of a seed mcdium of the same composition as that used inExample 1 and the inoculated medium was incubated under aeration andagitation at 36C for 24 hours. The resultant seed culture was furtherinoculated into lUUl. of a medium comprising l5il of D-glucose. 1.2% ofdry yeast. (L? of ammonium sulfate. 2.092 of CaCO;,. ltll) mg./l. ofL-phenylalanine and 50 mg/l. of L-tyrosine. 5U

mg/l. of L-tryptophan and 10 mg/l. of shikimic acid and incubated undersparging and stirring at 37C for 84 hours. whereupon 45 .5 g./liter ofD-ribose was accumulated in the medium.

The cells were removed from the broth and the filtrate was treated by aprocedure similar to that described in Example l. The procedure yielded3.5 kg. of crystalline D-ribose.

EXAMPLE 3 Bacillus pmm'lus No. 7l6 (lFO l3322) which was used in Example1 was inoculated into l()l. of a medium comprising 2% of sorbitol, 2% ofcorn steep liquor. 0.3% of dipotassium phosphate and 01% ofmonopotassium phosphate. and cultivated at 37C for 24 hours. Theresultant seed culture was used to inoculate 1001. of a mediumcomprising 15% of D-glucose. L09? of dry yeast. 0.5% of ammoniumsulfate, 2.0% of CaCO 5U mg./l. of L-tryptophan. 5O mg./l. ofL-tyrosine. mg/l. of Lphenylalanine and 500 mg./l. of maleic acid. Theinoculated medium was incubated under aeration and agitation at 37C forhours. by the end of which time the amount of Dribose accumulated was 60mg./ml. This broth was treated by a procedure similar to that describedin Example 1 to harvest 4.5 kg. of crystalline D-ribose.

EXAMPLE 4 The same mutant strain of Bacillus subrilis as that used inExample 2. No. 608 (lFO l3323). was inoculated into 10]. of a mediumcomprising 2% of sorbitol. 2% of corn steep liquor. 0.3% of dipotassiumphosphate and 0.1% of monopotassium phosphate. and cul tivated at 36Cfor 24 hours. The resultant seed culture was used to inoculate i001. ofa medium comprising 15)? of D-glucose. 2.0% of dry yeast. 0.5% of ammonium sulfate. 2.0% of CaCO and 750 mg./l. of maleic acid. The inoculatedmedium was incubated under aeration and agitation at 37C for 72 hours.whereupon 55.5 g./liter of D-ribose was accumulated in the medium.

The above fermentation broth was treated by a procedure similar to thatdescribed in Example l. The procedure yielded 4.3 kg. of crystallineD-ribose.

All strains used in the foregoing examples have been deposited atAmerican Type Culture Collection. Maryland. U.S.A. under the accessionnumbers listed below:

Strain Accession No.

Bmll/m mmllm No. 7 l6 ATCC Ill limillm \nlililix No 608 ATCC 1W5:

9 ism are L-tyrosine or its derivative and L-tryptophan or itsderivative and L-phenylalanine or its derivative.

3. A method according to claim I, wherein the factor which is nccessarfor growth of the microorganism is shikimic acid or a derivativethereof.

4. A method according to claim I, wherein the microorganism iscultivated in the culture medium at a temperature of from 25 to 45Cunder aerobic condi tion.

5. A method according to claim I, wherein a dibasic organic acid isadded to the medium for the cultivation of the microorganism to causethe microorganism to elaborate and accumulate D-ribose.

6. A method according to claim I, wherein the microorganism is a mutantof Bari/hm pumillus.

7. A method according to claim 6, wherein the microorganism is Bacilluspumillus No. 7l6l 8. A method according to claim I, wherein themicroorganism is Bacillus .s'ubrilis.

9. A method according to claim 8, wherein the microorganism is Bacillus.ru/nilix No. 608.

10. A method according to claim 5, wherein the dibasic organic acid isan aliphatic dibasic organic acid having 2 to 5 carbon atoms.

11. A method according to claim 10, wherein the aliphatic dibasicorganic acid is a member selected from the group consisting of malicacid. oxalic acid, maleic acid, fumaric acid. succinic acid, malonicacid glutaric acid and alkali salts thereof.

1. A METHOD PRODUCING D-RIBOSE, WHICH COMPRISES CULTIVATING AMICROORGANISM BELONGING TO THE GENUS BACILLUS IN A CULTURE MEDIUMCONTAINING ASSIMILABLE CARBON AND NITROGEN SOURCES AND FACTORS WHICH ARENECESSARY FOR GROWTH OF THE MICROOGANISMS, CAUSING SAID MICROORGANISM TOELABORATE AND ACCUMULATE D-RIBOSE, AND RECOVERING THE D-RIBOSE THUSACCUMULATED FROM THE RESULTANT CULTURE BROTH, THE TRANSKETOLASE ACTIVITYOF THE MICROORGANISM BEING LESS THAN 0.01 U M/LE/MIN./MG. PROTEIN.
 2. Amethod according to claim 1, wherein the factors which are necessary forgrowth of the microorganism are L-tyrosine or its derivative andL-tryptophan or its derivative and L-phenylalanine or its derivative. 3.A method according to claim 1, wherein the factor which is necessary forgrowth of the microorganism is shikimic acid or a derivative thereof. 4.A method according to claim 1, wherein the microorganism is cultivatedin the culture medium at a temperature of from 25* to 45*C under aerobiccondition.
 5. A method according to claim 1, wherein a dibasic organicacid is added to the medium for the cultivation of the microorganism tocause the microorganism to elaborate and accumulate D-ribose.
 6. Amethod according to claim 1, wherein the microorganism is a mutant ofBacillus pumillus.
 7. A method according to claim 6, wherein themicroorganism is Bacillus pumillus No.
 716. 8. A method according toclaim 1, wherein the microorganism is Bacillus subtilis.
 9. A methodaccording to claim 8, wherein the microorganism is Bacillus subtilis No.608.
 10. A method according to claim 5, wherein the dibasic organic acidis an aliphatic dibasic organic acid having 2 to 5 carbon atoms.
 11. Amethod according to claim 10, wherein the aliphatic dibasic organic acidis a member selected from the group consisting of malic acid, oxalicacid, maleic acid, fumaric acid, succinic acid, malonic acid, glutaricacid and alkali salts thereof.